Name: Valutazione Quantitativa Ottimizzata della Stabilità dellRNA Potenziatore nelle Cellule Staminali Embrionali di Topo (Video) | JoVE Journal
Uploaded: 2025-11-21T14:45:00.000+00:00
Duration: 3 min 34 s
Description: Scopri come misurare la stabilità degli eRNA nelle cellule staminali embrionali di topo usando Actinomycin D, RT-qPCR e analisi di regressione non lineare. Questo metodo quantitativo consente di modellare con precisione la dinamica del decadimento degli enhancer RNA.

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<li>Cinghu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intragenic+enhancers+attenuate+host+gene+expression%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">Intragenic enhancers attenuate host gene expression.</a> Mol Cell. 68 (1), 104-117.e106 (2017).</li>
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<li>Sohni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+switching+of+active+promoter+and+enhancer+domains+regulates+Tet1+and+Tet2+expression+during+cell+state+transitions+between+pluripotency+and+differentiation%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Dynamic switching of active promoter and enhancer domains regulates Tet1 and Tet2 expression during cell state transitions between pluripotency and differentiation.</a> Mol Cell Biol. 35 (6), 1026-1042 (2015).</li>
<li>Borsari, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancers+with+tissue-specific+activity+are+enriched+in+intronic+regions%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Enhancers with tissue-specific activity are enriched in intronic regions.</a> Genome Res. 31 (8), 1325-1336 (2021).</li>
<li>Bressin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-sensitive+nascent+transcript+sequencing+reveals+BRD4-specific+control+of+widespread+enhancer+and+target+gene+transcription%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">High-sensitive nascent transcript sequencing reveals BRD4-specific control of widespread enhancer and target gene transcription.</a> Nat Commun. 14 (1), 4971(2023).</li>
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<li>Rada-Iglesias, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+unique+chromatin+signature+uncovers+early+developmental+enhancers+in+humans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A unique chromatin signature uncovers early developmental enhancers in humans.</a> Nature. 470 (7333), 279-283 (2011).</li>
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<li>Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+the+embryonic+stem+cell+state%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Control of the embryonic stem cell state.</a> Cell. 144 (6), 940-954 (2011).</li>
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</ol>

<table><tbody><tr><td>2-mercaptoetanolo</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>Actinomicina D</td><td>Sigma-Aldrich</td><td>A9415</td><td></td></tr><tr><td>cloroformio</td><td>Sostanze chimiche Daejung</td><td>2548-4105</td><td></td></tr><tr><td>DMSO</td><td>GenDEPOT</td><td>D2680</td><td></td></tr><tr><td>Siero fetale bovino (FBS)</td><td>Sigma-Aldrich</td><td>F0392</td><td></td></tr><tr><td>Gelatina</td><td>Sigma-Aldrich</td><td>G1890-100G</td><td></td></tr><tr><td>Glasgow' s Medium Essenziale Minimo (GMEM)</td><td>Gibco</td><td>11710035</td><td></td></tr><tr><td>GraphPad Prism (versione 9)</td><td>GraphPad Software</td><td></td><td></td></tr><tr><td>Isopropanolo</td><td>Sostanze chimiche Daejung</td><td>5035-4105</td><td></td></tr><tr><td>Sostituzione del siero KnockOut (KOSR)</td><td>Gibco,&nbsp;</td><td>10828028</td><td></td></tr><tr><td>Fattore inibitore della leucemia (LIF)</td><td>Merck</td><td>ESG1106</td><td></td></tr><tr><td>Soluzione di Amminoacidi Non Essenziali (MnEA)</td><td>Welgene</td><td>LS005</td><td></td></tr><tr><td>Kit di sintesi cDNA M-MLV &nbsp;</td><td>Enzimomica</td><td>EZ006S</td><td></td></tr><tr><td>OPTIZEN NanoQ Plus&nbsp;</td><td>KLAB</td><td>01-QI685-M-16-C</td><td></td></tr><tr><td>Penicillina-streptomicina</td><td>Welgene</td><td>LS202-02</td><td></td></tr><tr><td>Power SYBR-Green PCR master mix&nbsp;</td><td>Biosistemi applicati</td><td>4368708</td><td></td></tr><tr><td>QuanStudio1 Sistema PCR in tempo reale&nbsp;</td><td>Biosistemi applicati</td><td>A40425</td><td></td></tr><tr><td>Piruvato di sodio</td><td>Welgene</td><td>LS013</td><td></td></tr><tr><td>TRIzol&nbsp;</td><td>Invitrogen</td><td>15596018</td><td></td></tr></tbody></table>

valutazione quantitativa ottimizzata della stabilità dell'rna potenziatore nelle cellule staminali embrionali di topo

Optimized Quantitative Assessment of Enhancer RNA Stability in Mouse Embryonic Stem Cells

Valutazione Quantitativa Ottimizzata della Stabilità dell'RNA Potenziatore nelle Cellule Staminali Embrionali di Topo

Enhancer RNAs (eRNAs) are non-coding transcripts produced from transcriptionally active enhancer regions and are increasingly recognized as key regulators ...

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Oct4GiP Reporter Assay per studiare i geni che regolano il mouse sulle cellule staminali embrionali di manutenzione e di auto-rinnovamento

Singolo gene delle cellule saggio di espressione è necessaria per comprendere eterogeneità di cellule staminali.

Profiling cellule staminali embrionali umane individuale mediante RT-PCR

La convalida sperimentale dell'attività dell'enhancer è meglio affrontata dall'analisi della perdita di funzione. Viene presentato un protocollo efficiente che utilizza la delezione mediata da CRISPR/Cas9 per studiare la regolazione allele-specifica della trascrizione genica nelle cellule ES F1 che contengono un genoma ibrido (Mus musculus129 x Mus castaneus).

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Analisi quantitativa di espressione della proteina per studiare Lineage specifica nel topo preimpianto embrioni

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Previsione e Validazione del gene regolatore elementi attivato durante retinoico Acid Induced embrionali cellule staminali Differenziazione

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Questo protocollo descrive un nuovo test di enhancer-reporter transitorio basato su rAAV. Questo test può essere utilizzato per indurre l'espressione guidata dal potenziatore in vivo nel cervello del topo.

Implementazione AAV di costrutti di espressione basati su enhancer in vivo nel cervello di topo

Questo protocollo fornisce una pipeline computazionale semplificata per quantificare le trascrizioni dei potenziatori nascenti. Integrando l'accessibilità della cromatina, le caratteristiche della cromatina e i dati trascrizionali, consente un rilevamento accurato e un'analisi specifica del filamento dell'attività dell'enhancer in regioni intrageniche complesse, pur rimanendo accessibile ai ricercatori senza un'ampia formazione in bioinformatica.

Una pipeline computazionale per la quantificazione dell'RNA dell'enhancer intergenico/intragenico nelle cellule staminali embrionali di topo

Scopri come misurare la stabilità degli eRNA nelle cellule staminali embrionali di topo usando Actinomycin D, RT-qPCR e analisi di regressione non lineare. Questo metodo quantitativo consente di modellare con precisione la dinamica del decadimento degli enhancer RNA.

Valutazione Quantitativa Ottimizzata della Stabilità dell'RNA Potenziatore nelle Cellule Staminali Embrionali di Topo

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